CN100371453C - Puerarin hydroxylation microbial transformation method - Google Patents
Puerarin hydroxylation microbial transformation method Download PDFInfo
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- CN100371453C CN100371453C CNB2006100385461A CN200610038546A CN100371453C CN 100371453 C CN100371453 C CN 100371453C CN B2006100385461 A CNB2006100385461 A CN B2006100385461A CN 200610038546 A CN200610038546 A CN 200610038546A CN 100371453 C CN100371453 C CN 100371453C
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- HKEAFJYKMMKDOR-VPRICQMDSA-N puerarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 HKEAFJYKMMKDOR-VPRICQMDSA-N 0.000 title claims abstract description 44
- 230000000813 microbial effect Effects 0.000 title claims abstract description 18
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- GARZMRVWLORUSR-VPRICQMDSA-N 3-(3,4-dihydroxyphenyl)-7-hydroxy-8-[(2s,3r,4r,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]chromen-4-one Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=CC(C2=O)=C1OC=C2C1=CC=C(O)C(O)=C1 GARZMRVWLORUSR-VPRICQMDSA-N 0.000 abstract description 18
- DRVXWCPMNIEOLC-UHFFFAOYSA-N 3'-hydroxypuerarin Natural products OCC1OC(Oc2c(O)cc(O)c3C(=O)C(=COc23)c4ccc(O)c(O)c4)C(O)C(O)C1O DRVXWCPMNIEOLC-UHFFFAOYSA-N 0.000 abstract description 14
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Abstract
The present invention relates to a microbial conversion method for hydroxylating puerarin. The present invention comprises the following steps: inoculating separated and preserved microbial strains with the activity of puerarin hydroxylase into cultivating liquid for fermentation and culture; separating the fermentation liquid from mycelia after the fermentation is finished; using the obtained mycelia as resting cells to add to conversion liquid containing puerarin to convert the puerarin into hydroxy puerarin; removing the mycelia after the conversion is finished; treating supernatant liquid by concentration and low-temperature drying to obtain a 3'-hydroxy puerarin solid; or extracting the supernatant liquid of the conversion liquid by an organic solvent; carrying out back extraction from an organic phase by water; obtaining a solution containing the 3-hydroxy puerarin from the obtained aqueous-phase extraction liquid by concentration and silica gel chromatography and separation; then concentrating the solution containing the 3-hydroxy puerarin or treating the solution containing the 3-hydroxy puerarin at a low temperature to obtain 3-hydroxy puerarin powder or crystals.
Description
Technical field
The present invention relates to a kind of puerarin hydroxylation microbial transformation method.
Technical background
Puerarin (Puerarin) chemistry is by name: 4 ', and 7-dihydroxyl-8-β-D-glucose isoflavones.Its toxic side effect is little, and physiological function is more widely arranged: as have anti-arrhythmia and hypotensive; Coronary artery dilator, the protection myocardial ultrastructure reduces myocardial infarct size; Expansion of cerebral vascular, the cerebral blood flow increasing amount is improved big cerebral oxygen supply; Improve organ microcirculations such as ear, retina; Anticoagulant improves hemorheology, and the effect of reduction blood viscosity and low-density lipoprotein are from the degree of oxidized damage; Can remove free radical and antioxygenation; Protection arteries endotheliocyte stops atherosclerosis, prevents thrombosis; Effect than the demulcent lowering blood glucose is arranged; The diabetes rat renal function had provide protection.Sato (1992) report 3 '-the hydroxyl root of kudzu vine have good antioxidant activity; And Cao (1999) report 3 '-the hydroxyl puerarin only is 1/tens of a puerarin at the intravital content of plant.
Summary of the invention
The object of the invention is to provide a kind of puerarin hydroxylation microbial transformation method: but screening obtain catalysis Puerarin be 3 ' hydroxyl-puerarin (3 '-hydroxypuerarin) microorganism strains, so that adopt the method for microbial transformation, acquisition 3 '-the hydroxypuerarin product.The molecular structural formula of Puerarin and 3 '-hydroxypuerarin is seen formula 1.
R1 R2 R3 R4
Puerarin glc H H H
3’-hydroxylpuerarin?glc H H OH
The molecular structural formula of formula 1.Puerarin and 3 '-hydroxypuerarin
The present invention adopts the microbial strains with puerarin hydroxylase vigor of separation and preservation to be inoculated in the nutrient solution, carries out fermentation culture; After the fermentation ends, fermented liquid is separated with mycelium; Institute's mycelium that obtains joins in the conversion fluid that contains puerarin as resting cell, makes puerarin be converted into the hydroxyl puerarin; After transform finishing, remove mycelium, supernatant liquor through concentrate, after the cryodrying processing, can get 3 '-hydroxyl puerarin solid; Or with the supernatant liquor organic solvent extraction of conversion fluid, water is stripped from organic phase again, the water extraction liquid that obtains separates through concentrated, silica gel column chromatography, can obtain containing 3 '-solution of hydroxyl puerarin, to contain 3 again '-solution of hydroxyl puerarin through concentrate or subzero treatment after, can obtain 3 '-hydroxyl puerarin powder or crystal.
Above-mentioned microbial strains with puerarin hydroxylase vigor can be a trichoderma harziarum, but also can be the microorganism or the mixing microorganisms of any hydroxylation puerarin.Said trichoderma harziarum can be Trichoderma harzianum ATCC20671, Trichoderma harzianum ATCC64060, Trichoderma harzianum ATCC60850, Trichoderma harzianum ATCC52443, Trichoderma harzianum ATCC52444, one or more among the Hypocrea lixii ATCC MYA-2453 etc.The bacterial strain of the plan name Trichoderma harzianum NJ01 of contriver's screening, be used for the present invention better effect is arranged, this bacterial strain has been stored in Pekinese China Committee for Culture Collection of Microorganisms common micro-organisms center on November 1st, 2005, preservation CGMCC NO.1523, classification called after trichoderma harziarum Trichoderma harzianum.
The above-mentioned nutritive substance that is used for the nutrient solution of fermentation culture has no particular limits, it can be any nutrient media that is suitable for microorganism growth, as the PDA nutrient solution, czapek's solution, or any be carbon source with starch, Semen Maydis powder, molasses, glucose, sucrose etc., with corn steep liquor, soybean cake powder, groundnut meal, cottonseed meal etc. are the substratum of nitrogenous source.
Above-mentioned puerarin hydroxylation microbial fermentation culture condition is: 20～40 ℃, 10～400rrpm shakes bottle vibration ventilation or stirs ventilation or pipeline ventilation oxygen-supplying, 12～96 hours reaction times.
Above-mentioned fermented liquid separates with mycelium, can adopt and filter or centrifugal separation method acquisition mycelium.
Above-mentioned puerarin hydroxylation conversion of resting cells reaction can be carried out in water, organic phase or water-organic two-phase.The water conversion condition is: the substrate quality than concentration for greater than 0.01% to saturated, in the buffered soln of the slant acidity of any one pH3～7; Organic phase is ethanol, dimethyl sulfoxide (DMSO), dimethyl formamide, or the less organic solvent of other pair cell toxicity; Water-organic phase is the arbitrary proportion mixture of above-mentioned water and organic phase.The invert point scope is at 20～40 ℃, 10～400rpm shakes bottle vibration ventilation or stirs ventilation or pipeline ventilation oxygen-supplying, and 12～96 hours reaction times is after conversion finishes, with conversion fluid and thalline by centrifugal or filter and separate, can obtain to contain 3 '-conversion fluid of hydroxypuerarin.
Above-mentionedly contain 3 '-hydroxypuerarin solution through concentrate, after cryodrying handles, can get 3 '-hydroxyl puerarin solid (purity 〉=50%); But or to above-mentionedly contain 3 '-add the organic solvent of propyl carbinol or any other extraction product in the hydroxypuerarin solution, vibration, extraction, leave standstill or centrifugal layering after, the water extraction again of organic phase part, extraction liquid separates through concentrated, silica gel column chromatography, can obtain containing 3 '-solution of hydroxypuerarin, again through concentrate or subzero treatment after, can obtain 3 '-hydroxypuerarin powder (purity 〉=90%).Make 3 '-hydroxypuerarin is in the DMSO
1H-NMR and
13The analysis result of the chemical shift of C-NMR is as follows:
1H-NMR(400MH，DMSO-d
6+D
2O)：
δ(ppm)＝4.81(1H，d，J＝9.6Hz，glcH-1)，6.76(1H，d，J＝8.2Hz，H-5′)，6.80(1H，dd，J＝8.1，1.9Hz，H-6′)，7.03(1H，d，J＝1.8Hz，H-2′)，6.99(1H，d，J＝8.8Hz，H-6)，7.93(1H，d，J＝8.8Hz，H-5)，8.30(1H，s，H-2)。
13C-NMR(100MHz，DMSO-d
6)：
δ(ppm)＝153.0(C-2)，123.7(C-3)，175.3(C-4)，126.7(C-5)，115.8(C-6)，161.5(C-7)，113.1(C-8)，153.0(C-9)，117.4(C-10)；123.4(C-1′)，115.8(C-2′)，145.2(C-3′)，145.7(C-4′)，117.1(C-5′)，120.2(C-6′)；73.9(C-1″)，71.3(C-2″3，79.2(C-3″)，70.9(C-4″，82.3(C-5″)，61.9(C-6″)。
MS analyze make 3 '-molecular weight of hydroxypuerarin is 432.
The present invention with microbial conversion process obtain 3 '-its anti-oxidant activity of hydroxylation puerarin is higher 20 times than puerarin, and water-soluble is more than 1.3 of substrate, and other pharmacologically active all has improvement in various degree, and technology is simple, the product purity height has good application prospects.
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
Embodiment 1. puts into the 500ml triangular flask with 100ml PDA substratum, behind 121 ℃ of high pressure steam sterilizations, insert Trichoderma harzianum NJ01 (CGMCC1523) bacterial classification, 30 ℃, after the 200rpm oscillation and fermentation cultivation 36 hours, stop fermentation, filtered through gauze separation of mycelial and fermented liquid, the mycelium of getting 62.5ml bacterium liquid join and contain 0.5% puerarin, in the 250ml triangular flask of 25ml phosphate buffered saline buffer (pH5.3), 30 ℃, the reaction of puerarin hydroxylation conversion of resting cells is carried out in 200rpm vibration ventilation, after 48 hours, stops conversion reaction, filter to remove mycelium, contain in the supernatant liquor 38% 3 '-hydroxypuerarin.Supernatant liquor with twice of 5ml n-butanol extraction after, 3 '-hydroxypuerarin content can reach 80%.Concentrated solution after silica gel column chromatography separate to concentrate, obtain purity reach 95% 3 '-the hydroxypuerarin powder.
Embodiment 2. puts into the 250ml triangular flask with 50ml PDA substratum, behind 121 ℃ of high pressure steam sterilizations, insert Trichoderma harzianum ATCC20671 bacterial classification, 20 ℃, the 150rpm oscillation and fermentation cultivation, after 96 hours, stop fermentation, filtration makes mycelium separate with fermented liquid, the mycelium of getting 62.5ml bacterium liquid joins and contains 0.5% puerarin, in the 250ml triangular flask of 25ml phosphate buffered saline buffer (pH5.3), 20 ℃, the reaction of puerarin hydroxylation conversion of resting cells is carried out in 150rpm vibration ventilation, after 12 hours, stop conversion reaction, filter to remove mycelium, contain in the supernatant liquor (purity 32%) 3 '-hydroxypuerarin.Supernatant liquor with twice of 10ml n-butanol extraction after, 3 '-hydroxypuerarin purity can reach 80%.After concentrating chromatographic separation, can obtain (purity 90%) 3 '-the hydroxypuerarin powder.
Embodiment 3. puts into the 5L automatic fermenter with 3.5L PDA substratum, behind 121 ℃ of high pressure steam sterilizations, insert Trichoderma harzianum ATCC64060 bacterial classification, 28 ℃, the 400rpm aerobic fermentation, after 36 hours, stop fermentation, filtration makes mycelium separate with fermented liquid, the mycelium of getting 62.5ml bacterium liquid joins and contains 0.5% puerarin, in the 250ml triangular flask of 25ml phosphate buffered saline buffer (pH5.3), 30 ℃, the reaction of puerarin hydroxylation conversion of resting cells is carried out in 200rpm vibration ventilation, after 48 hours, stop conversion reaction, filter to remove mycelium, contain in the supernatant liquor 45% 3 '-hydroxypuerarin.Supernatant liquor with twice of 15ml n-butanol extraction after, 3 '-hydroxypuerarin content can reach 80%.After concentrating chromatographic separation, can obtain (purity 90%) 3 '-the hydroxypuerarin powder.
Embodiment 4. contains (2%) starch with 1 00ml, (2%) the corn steep liquor substratum is put into the 500ml triangular flask, behind 121 ℃ of high pressure steam sterilizations, insert Trichoderma harzianum NJ01 (CGMCC1523) bacterial classification, 28 ℃, the 250rpm oscillation and fermentation cultivation, after 36 hours, stop fermentation, filtration makes mycelium separate with fermented liquid, and the mycelium of getting 62.5ml bacterium liquid joins and contains 0.5% puerarin, in the 250ml triangular flask of 25ml phosphate buffered saline buffer (pH5.5), 28 ℃, the reaction of puerarin hydroxylation conversion of resting cells is carried out in 250rpm vibration ventilation.After 48 hours, stop conversion reaction, filter to remove mycelium, supernatant liquor directly concentrates, after the drying, can obtain 3 '-hydroxypuerarin powder (purity is 50%).
Embodiment 5. contains 100ml (1%) starch, (3%) the corn steep liquor substratum is put into totally 15 bottles of 500m1 triangular flasks, behind 121 ℃ of high pressure steam sterilizations, insert the spore of Trichoderma harzianum NJ01 (CGMCC1523) bacterial strain, 25 ℃, the 220rpm aerobic fermentation, after 36 hours, stop fermentation, filtration makes mycelium separate with fermented liquid, the mycelium of getting 1500ml bacterium liquid joins 475ml phosphate buffered saline buffer (pH5.3), adds the dimethyl sulphoxide solution (containing puerarin 2g/L in the mixed phase) that 25ml contains the 40g/L puerarin again and divides in 5 the 500ml triangular flasks of packing into.Above-mentioned mixed phase is in 25 ℃, the reaction of puerarin hydroxylation conversion of resting cells is carried out in 200rpm vibration ventilation, after 72 hours, stop conversion reaction, filter to remove mycelium, supernatant liquor with twice of 250ml n-butanol extraction after, separate through the HPLC preparative chromatography, behind the condensing crystal, can obtain 0.6g purity and be 98% 3 '-the hydroxypuerarin powder.
Embodiment 6: substantially the same manner as Example 1, but bacterial classification adopts Trichoderma harzianumATCC60850.
Embodiment 7: substantially the same manner as Example 1, but bacterial classification adopts Trichoderma harzianumATCC52443.
Embodiment 8: substantially the same manner as Example 1, but bacterial classification adopts Hypocrea lixii ATCCMYA-2453.
The invention is not restricted to these disclosed embodiments, the present invention will cover the scope described in the patent book, and the various modification of claim scope and equivalence variation.
Claims (5)
1. puerarin hydroxylation microbial transformation method, described method is:
The microbial strains that will have puerarin hydroxylase vigor is inoculated in the nutrient solution, and 20～40 ℃, 10～400rpm shakes bottle vibration ventilation or stirs ventilation or pipeline ventilation oxygen-supplying, ferments 12～96 hours; After the fermentation ends, fermented liquid is separated with mycelium; Institute's mycelium that obtains joins in the conversion fluid that contains puerarin as resting cell, makes puerarin be converted into the hydroxyl puerarin; Wherein said microbial strains with puerarin hydroxylation vigor is a trichoderma harziarum; Wherein said conversion is to carry out in water, organic phase or water-organic two-phase:
The water conversion condition is: the substrate quality than concentration for greater than 0.01% to saturated, in the buffered soln of the slant acidity of any one pH 3～7;
Organic phase is ethanol, dimethyl sulfoxide (DMSO), dimethyl formamide, or the less organic solvent of other pair cell toxicity;
Water-organic phase is the mixture of above-mentioned water and organic phase;
The invert point scope is at 20～40 ℃, and 10～400rpm shakes bottle vibration ventilation or stirs ventilation or pipeline ventilation oxygen-supplying, 12～96 hours reaction times;
After transform finishing, remove mycelium, supernatant liquor through concentrate, after the cryodrying processing, can get 3 '-hydroxyl puerarin solid; Perhaps the supernatant liquor of conversion fluid is handled with organic solvent extraction, organic solvent extraction liquid is after water is stripped again, separate through concentrated, silica gel column chromatography, obtain containing 3 '-solution of hydroxyl puerarin, to contain 3 again '-solution of hydroxyl puerarin through concentrate or subzero treatment after, obtain 3 '-hydroxyl puerarin powder or crystal.
2. puerarin hydroxylation microbial transformation method according to claim 1, it is characterized in that, described trichoderma harziarum is Trichoderma harzianum CGMCC1523, Trichoderma harzianum ATCC20671, Trichoderma harzianum ATCC64060, Trichoderma harzianum ATCC60850, Trichoderma harzianumATCC52443, one or more among the Hypocrea lixii ATCC MYA-2453.
3. puerarin hydroxylation microbial transformation method according to claim 1 and 2 is characterized in that the described nutrient solution that is used for fermentation culture, is the PDA nutrient solution.
4. puerarin hydroxylation microbial transformation method according to claim 3 is characterized in that fermentation culture separates with mycelium, adopts filter separation method or centrifugal separation method.
5. puerarin hydroxylation microbial transformation method according to claim 4, the organic solvent that it is characterized in that said extraction conversion fluid is a propyl carbinol.
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